Marginal Zone B Cells Facilitate CD4 T Cell Proliferation in Response to Factor VIII in Mice with Hemophilia Α

BACKGROUND: The development of anti-factor VIII (FVIII) antibodies represents a significant barrier to FVIII replacement therapy in patients with hemophilia A. Despite this, the immune factors that regulate anti-FVIII antibody formation remain incompletely understood.

We have previously shown that shortly after injection, FVIII co-localizes with marginal zone (MZ) B cells in the marginal sinus of the spleen in mice with hemophilia A. Moreover, mice depleted of MZ B cells did not produce detectable anti-FVIII IgG antibodies. Given the ability of MZ B cells to trap and rapidly respond to blood-borne antigens, produce both IgM and IgG antibodies and activate CD4 T cells, while also trafficking antigen to B cell follicles, these results suggest that MZ B cells likely play a critical role in the initiation and subsequent orchestration of an immune response to FVIII.

In contrast to other model antigens, anti-FVIII antibody formation occurs only following multiple FVIII exposures in both human and mouse models. As CD4 T cells facilitate long-lasting antibody formation, and have previously been shown to play an important role in anti-FVIII antibody development (Bray GL, et al. Am J Hematol 1993; Reding MT, et al. Thromb Haemost 2000), it is possible that each FVIII exposure event eventually propagates a CD4 T cell response to a threshold necessary to generate an optimal antibody response. Consistent with this, we have previously shown that FVIII-specific CD4 T cell proliferation requires more than 1 prior exposure to FVIII. An improved understanding of the overall timing and factors influencing CD4 T cell activation in the immune response to FVIII is necessary for the development of strategies to prevent antibody formation.

OBJECTIVE: Define the key factors that regulate CD4T cell activation following FVIII exposure and the number of exposure events required to observe FVIII-specific CD4 T cell proliferation.

METHODS: Hemophilia A mice +/- CD4T cell depletion or MZ B cell depletion or S1PR1-TSS mice were challenged with 1-4 weekly exposures to FVIII. Anti-FVIII IgM and IgG was examined in plasma by enzyme-linked immunoassay (ELISA). To assess FVIII-specific CD4T cell proliferation, we engineered FVIII-OVA, which contains the ovalbumin peptide recognized by OTII T cell receptor transgenic T cells. CFSE-labeled OTII splenocytes were adoptively transferred into mice with hemophilia A, mice lacking B cells and MZ B cell-depleted mice, followed by administration of FVIII-OVA.

RESULTS: Anti-FVIII IgG formation is CD4T cell-dependent, however IgM is readily produced in the absence of CD4T cells. In contrast, MZ B cell depletion prevented the formation of both IgM and IgG antibodies against FVIII. FVIII is unable to cause proliferation of CD4T cells or produce IgG antibodies following initial exposure, but instead significantly increases following the 3^rd exposure to FVIII. Mapping the kinetics of antibody formation has uncovered that anti-FVIII IgM formation peaks and anti-FVIII IgG formation begins after the 3^rd FVIII exposure, at the time of CD4 T cell proliferation. We next examined the influence of B cells on CD4 T cell proliferation, and observed that FVIII-specific CD4T cell proliferation is significantly impaired in B cell-deficient recipients. Moreover, in mice who have undergone MZ B cell depletion, there is likewise an absence of anti-FVIII IgM and IgG and a lack of measurable CD4 T cell proliferation. Finally, in S1PR1-TSS mice with MZ B cells that are unable to traffic antigen to the B cell follicle, anti-FVIII IgM and IgG formation occurs at the same rate as control mice.

CONCLUSION: In summary, our findings suggest that MZ B cells are required for anti-FVIII IgM and IgG antibody formation as well as FVIII-specific CD4 T cell proliferation, which requires 3 antigen exposures. The finding that MZ B cells are not required to traffic antigen to the B cell follicle contributes to our hypothesis that MZ B cell production of IgM, with or without direct activation of CD4 T cells contributes to FVIII-specific CD4 T cell activation. These results suggest that targeted approaches designed to prevent anti-FVIII antibody formation may be most effectively achieved by defining key factors that regulate MZ B cell activation following initial FVIII exposure. In doing so, the present findings and future work will likely uncover important pathways to effectively target to reduce, or event prevent anti-FVIII antibody production.

Meeks:Pfizer: Consultancy; Spark: Consultancy; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: advisory board, Research Funding; Biomarin: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: advisory board; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: advisory board; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy, Other: advisory board; Shire: Consultancy, Other: advisory board; Octapharma: Research Funding. Stowell:Grifols: Consultancy; Cellics: Consultancy; Novartis: Consultancy; Alexion: Consultancy; Aregenx: Consultancy.